S-Phase Progression of North American ATLL Cells Is Critically Regulated By the Proto-Oncogene BCL6 and Targetable By PARP Inhibition

Adult T-cell leukemia/lymphoma (ATLL) is a disease of malignant T cells that develops in human T-lymphotropic virus-1 (HTLV-1) carriers. ATLL is one of the most aggressive form of NHL with an abysmal survival and no promising targeted therapies. We and others have shown that ATLLs diagnosed in the Japanese (J-ATLL) and North American (NA-ATLL) patients have very different clinical behavior, with the NA-ATLL variant showing a more aggressive clinical course and carrying more frequent epigenetic mutations compared to J-ATLLs. The most frequently mutated epigenetic modifier gene in NA-ATLL is EP300 which is mutated in 20% of our patients compared to only 5.7% of J-ATLLs.

EP300 encodes p300, a lysine acetyltransferase with a multitude of protein substrates including histone H3 and a number of important transcription factors, among which are p53 and BCL6. Previously, we have demonstrated that p300 mutations correlate with reduced p53 levels and compromised p53 transcriptional activity. In this study, we discovered for the first time that the B cell lymphoma oncogene BCL6 is expressed in primary ATLL samples and in vitro cultured ATLL cells. As expected, p300 mutation corresponds to reduced BCL6 acetylation. More importantly, notable differences in BCL6-regulated transcriptome were uncovered between EP300 WT and mutated samples based on RNA-seq analysis. Functional importance of BCL6 was then investigated using the siRNA-based knock-down approach in combination with a small compound BCL6 inhibitor, FX1. Unexpectedly, we discovered that nearly all cultured NA-ATLL samples are very sensitive to BCL6 inactivation (FX1 IC50 = 36+/-14 uM), while the 6 J-ATLL cell lines tested showed a wide range of FX1 sensitivity. This disparity is also seen in the pattern of BCL6-regulated genes. Specifically, based on clustering analysis, BCL6-regulated Reactome Pathways are more closely related among 4 NA-ATLL cell lines relative to those among 4 J-ATLL cell lines.

Finally, we demonstrate that continued expression of BCL6 is critical for the survival of NA-ATLL cells going through the S-phase of cell cycle since the S-phase fraction was selectively depleted following BCL6 inactivation by either BCL6 siRNA transfection or FX1 treatment. Prompted by the recognition that the S-phase could be an "achilles heel" of NA-ATLL cells, we tested the sensitivity of a number of NA-ATLL samples (both primary and cell lines) and J-ATLL cell lines to 3 S-phase directed drugs: the PARP inhibitor, Olaparib, the Wee1 inhibitor, AZD-1775, and the ATR inhibitor, AZD-6738. While virtually all ATLL samples tested showed sensitivity to the Wee1 (IC50 = 0.2 to 1.1 uM) and ATR (IC50 = 1.0 to 14.5 uM) inhibitors, Olaparib sensitivity demonstrated ethnic and genotype specificity. Specifically, except the one and only EP300-mutated sample, 5/6 J-ATLL cell lines are resistant to Olaparib. In comparison, among the 10 NA-ATLL samples tested, 6/10 are Olaparib sensitive (IC50 =20-40 uM). The 4 Olaparib resistant samples carried inactivating mutations in either TP53 or RICTOR/PIK3CD.

In summary, this study provides additional insights into the functional consequences of EP300 mutations in NA-ATLL. For the first time, we demonstrate that BCL6 is expressed in NA-ATLL and is critically required for survival of these cells when they transit through the S-phase of cell cycle. Furthermore, our discovery that NA-ATLLs are often sensitive to Olaparib treatment in vitro warrants follow up investigation that could lead to a novel targeted therapeutic strategy for this devastating malignancy.